Assays of samples (i.e. biological, environmental etc) are routinely used to detect and measure the presence and the concentration of analytes such as drugs, pollutants, chemicals, contaminants, or the like. Regardless of the format for the assays, the analyte concentration is inferred from the dose-response curve. This can be done by finding the ordinate on the dose-response curve corresponding to the signal for the unknown concentration of analyte in the sample. The later quantity is given by the value of the abscissa. The dose-response curve is typically non-linear and it can be prepared by assaying standard samples containing known concentrations of the analyte. The number of standard samples and their concentrations are selected in order to determine the analyte concentrations with sufficient accuracy over the expected assay range.
A variation in the assay conditions, however, can modify the actual relationship between the measured signal and analyte concentration from that predicted by the initial dose-response curve. It is therefore often necessary for an analyzer to accurately control and maintain variables that affect the dose-response curve. One of the most common variables that produces significant variations in an assay signal is temperature. Changes in temperature lead to assay variations due to the strong thermal dependence of reaction rates and assay kinetics. For example, it is well known that the signal of a homogeneous enzyme assay, in which an enzyme is acting on a substrate to produce a measurable signal, has a thermal dependence of approximately 4% per degree Celsius.
In the past, automated analyzer systems have included complex thermal stabilization systems to ensure a constant and stable temperature during the analytic phase of an assay. For example U.S. Pat. No. 4,483,823A (Umetsu et al., filed 1982) describes an automated analyzer in which a water bath with precise temperature control is employed to regulate the temperature of a sample vessel. In another example of the prior art, U.S. Pat. No. 4,933,146 A (Meyer and Greene, filed 1986) discloses a thermal subsystem for the accurate thermal regulation of a set of cuvettes in an automated analyzer. An alternative method to avoid degradation in assay performance due to thermal sensitivity is to measure a full set of calibrators and obtain a new dose-response curve each time samples are analyzed. In such a scheme, it is only important to keep the temperature stable during the assay. Unfortunately, this method is not practical since most assays require many separate calibrators for proper calibration. Running several calibrators each time samples are assayed leads to significantly impaired throughput, increased costs and added complexity, especially when many analytes are analyzed in parallel.
An attempt to solve the problems associated with assaying multiple calibrators involves the use of internal multiplexed calibrators. Instead of assaying physically separate calibrators, in which standards containing known concentrations of single or multiple analytes are assayed in the same manner as samples, assay reagents are designed to give, in addition to a principal signal indicative of the analyte concentration, a calibration signal to correct for variations in the assay parameters.
U.S. Pat. No. 5,648,274 (issued to Chandler et al.) describes the use of a single internal calibrator in a comparative dual assay and U.S. Pat. No. 5,387,503 (issued to Selmer et al.) describes the use of an internal calibration by the addition of foreign analytes to samples and detection of both the target and foreign analytes at separate areas on a solid support. In a variation of these methods, the sequential addition to the assay reagents of a sample followed by the internal calibrator is described in U.S. Pat. No. 6,514,770 (issued to Sorin). The disadvantage of using an internal calibrator is that insufficient calibration measurements are obtained to properly re-calibrate the initial dose-response curve. Additional disadvantages include the requirement for dedicated reagents, which are not always commercially available, and an analyzer capable of detecting two signals. For these reasons, internal calibrators are not readily integrated into a commercial assay platform.
Recently, a general method of correcting for changes in an assay signal due to variations such a temperature was disclosed in copending U.S. patent application Ser. No. 11/072,651, filed Mar. 7, 2005 entitled “Correction for Temperature Dependence of Assays”. This method involves assaying one or more additional calibrators each time samples are analyzed. In a preferred embodiment of this invention, no additional calibrator is added to the reagents and the inherent signal from the reagents themselves is used for assay calibration. Although this method produces satisfactory results for many different assay formats, its accuracy is limited when used with assays that exhibit thermal sensitivity due to complex mechanisms such as binding kinetics.
What is therefore required is a reliable method of accurately compensating for thermal variations in an assay signal that does not significantly impair assay throughput and can be applied to a wide range of assay formats.